[1]张瑞萍,邱相君,苏兵,等.头蛋白shRNA和骨形态发生蛋白2的协同作用对骨髓间充质干细胞成骨能力的影响[J].中医正骨,2019,31(09):1-6.
　ZHANG Ruiping,QIU Xiangjun,SU Bing,et al.The synergistic effects of Noggin shRNA and bone morphogenetic protein 2 on osteogenic ability of bone marrow mesenchymal stem cells[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2019,31(09):1-6.
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头蛋白shRNA和骨形态发生蛋白2的协同作用对骨髓间充质干细胞成骨能力的影响()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第31卷
期数:
2019年09期
页码:
1-6
栏目:
基础研究
出版日期:
2019-09-20

文章信息/Info

Title:
The synergistic effects of Noggin shRNA and bone morphogenetic protein 2 on osteogenic ability of bone marrow mesenchymal stem cells
作者:
张瑞萍1; 邱相君2; 苏兵3; 郑路路1
(1.洛阳市第一人民医院,河南 洛阳 471002; 2.河南科技大学,河南 洛阳 471003; 3.Quater诊断实验室,纽约 布法罗 14200)
Author(s):
ZHANG Ruiping1; QIU Xiangjun2; SU Bing3; ZHENG Lulu1
1.The First People's Hospital of Luoyang,Luoyang 471002,Henan,China 2.Henan University of Science and Technology,Luoyang 471003,Henan,China 3.Quater diagnosis laboratory,Buffalo 14200,New York,America
关键词:
间质干细胞;  骨髓;  骨形态发生蛋白质2;  转染;  头蛋白
Keywords:
mesenchymal stem cells;  bone marrow;  bone morphogenetic protein 2;  transfection;  Noggin
摘要:
目的:观察头蛋白shRNA和骨形态发生蛋白2(bone morphogenetic protein 2,BMP-2)的协同作用对骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成骨能力的影响。方法:分离、培养兔BMSCs,分为头蛋白shRNA基因干扰组、BMP-2过表达组、双基因转染组。构建慢病毒头蛋白shRNA干扰载体和BMP-2过表达载体。头蛋白shRNA基因干扰组、BMP-2过表达组分别加入头蛋白shRNA基因干扰载体和BMP-2过表达载体,双基因转染组同时加入头蛋白shRNA基因干扰载体和BMP-2过表达载体,进行基因转染。基因转染成功后,采用荧光定量PCR检测3组BMSCs中头蛋白和BMP-2的mRNA相对表达量,采用茜素红染色和碱性磷酸酶染色检测3组BMSCs的诱导成骨能力。结果:①细胞培养和基因转染鉴定结果。流式细胞仪检测第2代培养细胞,CD44和CD29表达分别为61.3%和57.2%,CD34和CD45表达分别为3.6%和2.4%,符合BMSCs表型特征。基因转染72 h后,3组BMSCs的转染率为(92.5±2.5)%,基因转染成功。②头蛋白和BMP-2 mRNA表达检测结果。基因转染成功后,3组细胞中头蛋白mRNA相对表达量的组间总体比较,差异有统计学意义(0.343±0.124,1.040±0.269,0.123±0.057; F=22.390,P=0.002); BMP-2过表达组较头蛋白shRNA基因干扰组和双基因转染组高(P=0.016,P=0.005),头蛋白shRNA基因干扰组和双基因转染组的组间差异无统计学意义(P=0.054)。3组细胞中BMP-2 mRNA相对表达量的组间总体比较,差异有统计学意义(1.120±0.363,3.667±0.431,4.833±0.475; F=59.680,P=0.000)。双基因转染组较头蛋白shRNA基因干扰组和BMP-2过表达组高(P=0.000,P=0.035),BMP-2过表达组高于头蛋白shRNA基因干扰组(P=0.001)。③茜素红和碱性磷酸酶染色结果。茜素红染色可见胞浆边缘呈现不透光的矿化结节。3组细胞茜素红染色阳性率的组间总体比较,差异有统计学意义[(62.317±3.969)%,(39.267±2.170)%,(72.320±4.133)%,F=68.870,P=0.000]; 双基因转染组高于头蛋白shRNA基因干扰组和BMP-2过表达组(P=0.039,P=0.000),头蛋白shRNA基因干扰组高于BMP-2过表达组(P=0.001)。碱性磷酸酶染色细胞内呈现灰黑色颗粒或块状沉淀。3组细胞碱性磷酸酶染色阳性率组间总体比较,差异有统计学意义[(55.157±9.959)%,(35.353±6.494)%,(82.337±10.594)%,F=19.750,P=0.002]; 双基因转染组高于头蛋白shRNA基因干扰组和BMP-2过表达组(P=0.032,P=0.003),头蛋白shRNA基因干扰组高于BMP-2过表达组(P=0.045)。结论:头蛋白shRNA和BMP-2的协同作用可以强化BMSCs的成骨能力。
Abstract:
Objective:To observe the synergistic effects of Noggin shRNA and bone morphogenetic protein 2(BMP-2)on osteogenic ability of bone marrow mesenchymal stem cells(BMSCs).Methods:The BMSCs were isolated from the femoral bone marrow of rabbits and were cultured in vitro.The second-generation BMSCs were randomly divided into Noggin shRNA gene interference group,BMP-2 overexpression group and double-gene transfection group.The lentivirus-mediated Noggin shRNA interference vectors and BMP-2 overexpression vectors were constructed.The BMSCs in Noggin shRNA gene interference group and BMP-2 overexpression group were transfected with Noggin shRNA gene interference vectors and BMP-2 overexpression vectors respectively,and the BMSCs in double-gene transfection group were co-transfected with Noggin shRNA gene interference vectors and BMP-2 overexpression vectors.After successful gene transfection,the relative expression levels of Noggin mRNA and BMP-2 mRNA in BMSCs were detected by using fluorescent quantitative PCR method,and the ability of BMSCs to induce osteogenesis was detected by using alizarin red staining(ARS)and alkaline phosphatase(ALP)staining.Results:The second-generation BMSCs were detected by using flow cytometry,and the results showed that the expressions of CD44,CD29,CD34 and CD45 were 61.3%,57.2%,3.6% and 2.4% respectively,which were consistent with the phenotypic characteristics of BMSCs.After 72-hour gene transfection,the transfection rate of BMSCs was(92.5+/-2.5)% in the 3 groups.After successful gene transfection,there was statistical difference in the relative expression level of Noggin mRNA in BMSCs between the 3 groups in general(0.343+/-0.124,1.040+/-0.269,0.123+/-0.057; F=22.390,P=0.002).The relative expression level of Noggin mRNA in BMSCs was higher in BMP-2 overexpression group compared to Noggin shRNA gene interference group and double-gene transfection group(P=0.016,P=0.005),and there was no statistical difference in the relative expression level of Noggin mRNA in BMSCs between Noggin shRNA gene interference group and double-gene transfection group(P=0.054).There was statistical difference in the relative expression level of BMP-2 mRNA in BMSCs between the 3 groups in general(1.120+/-0.363,3.667+/-0.431,4.833+/-0.475; F=59.680,P=0.000).The relative expression level of BMP-2 mRNA in BMSCs was higher in double-gene transfection group compared to Noggin shRNA gene interference group and BMP-2 overexpression group(P=0.000,P=0.035),and was higher in BMP-2 overexpression group compared to Noggin shRNA gene interference group(P=0.001).The opaque mineralized nodules along the cytoplasmic margin were found through ARS.There was statistical difference in the positive rate of ARS for BMSCs between the 3 groups in general(62.317+/-3.969,39.267+/-2.170,72.320+/-4.133%,F=68.870,P=0.000).The positive rate of ARS for BMSCs was higher in double-gene transfection group compared to Noggin shRNA gene interference group and BMP-2 overexpression group(P=0.039,P=0.000),and was higher in Noggin shRNA gene interference group compared to BMP-2 overexpression group(P=0.001).The gray-black granules or block-shaped sediments were found within BMSCs through ALP staining.There was statistical difference in the positive rate of ALP staining for BMSCs between the 3 groups in general(55.157+/-9.959,35.353+/-6.494,82.337+/-10.594%,F=19.750,P=0.002).The positive rate of ALP staining for BMSCs was higher in double-gene transfection group compared to Noggin shRNA gene interference group and BMP-2 overexpression group(P=0.032,P=0.003),and was higher in Noggin shRNA gene interference group compared to BMP-2 overexpression group(P=0.045).Conclusion:The synergistic effects of Noggin shRNA and BMP-2 can enhance the osteogenic ability of BMSCs.

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备注/Memo

备注/Memo:
通讯作者:张瑞萍 E-mail:lyxiew@126.com(收稿日期:2019-08-01 本文编辑:杨雅)
更新日期/Last Update: 2019-09-15